Project Summary The brain consumes a tremendous amount of energy to fuel its normal functioning. Because neurons lack substantial energy reserves, the brain relies on an on-demand system, orchestrated by a multicellular aggregate?the neurovascular unit?consisting of neurons, astrocytes and vascular cells, to match local blood supply to neuronal energy demands. This use-dependent increase in local blood flow (functional hyperemia) is mediated by a process termed neurovascular coupling. Although significant progress has been made in understanding the essential role of localized synaptic glutamatergic signaling in this process, very little is currently known about the broader cellular and molecular mechanisms underlying the spatiotemporal coordination of local and global vascular responses within the unique cortical angioarchitecture. The overall goal of this proposal is to identify how local and global signaling pathways interact to control the distribution of blood flow in response to increased neuronal activity. We propose a model for activity-dependent allocation of cerebral blood flow that depends on the integration of three elements: local synaptic glutamatergic signaling, retrograde intercellular conduction, and global neuromodulatory projections. The central hypothesis of our proposal is that localized synaptic communication between neural and vascular cells in the neurovascular unit must be complemented by spatiotemporal coordination of global vascular reactivity through vascular gap-junctional communication and neuromodulatory serotonergic signaling to achieve optimal brain perfusion. To test this hypothesis, we will employ two-photon fluorescence imaging of the vasculature and Ca2+ dynamics in vivo in fully awake, behaving animals in conjunction with knockout strategies, genetically encoded biosensors, DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) and optogenetics. The latter two approaches are novel and powerful, as they provide the ability to control the activity of specific cell types using physiologically inert molecules and light, respectively, without affecting neighboring cell types. Furthermore, our recent advances allow us to use a fully awake mouse model in our in vivo investigation of the interaction of local and global signaling in controlling cerebral blood flow. This eliminates the need for anesthetics, which have dramatic side effects on brain and blood dynamics. The goal of Aim 1 is to determine the contribution of the endothelium to conducted vasodilation initiated at the neurovascular unit. We will test the hypothesis that the endothelium, and not smooth muscle cells, mediates the conduction of vascular responses initiated at parenchymal vessels to upstream pial vessels, and that this process is critical for functional hyperemia during neurovascular coupling in vivo. The goal of Aim 2 is to elucidate the role of serotonin in controlling cerebral blood flow during neurovascular coupling. We will test the hypothesis that long-range neuromodulatory serotonergic signaling reflecting alertness status elicits vasomotor responses associated with functional hyperemia during neurovascular coupling in vivo. The goal of Aim 3 is to identify serotonin signaling pathway(s) in the cerebral microcirculation. We will test the hypothesis that serotonin initiates signaling pathways in different cell types, including smooth muscle cells, endothelial cells, astrocytes and interneurons, and that serotonin-mediated vascular responses are cell-type specific. Our investigations of this conceptual novel model may reveal new physiological processes essential to cerebral blood flow regulation and, ultimately, brain health.